There has been significant progress made in the understanding of T cell recognition of human tumor antigens, however the properties that enable T cells to mediate clinical cancer regression remain unclear. We hypothesized that an iterative translational strategy involving isolation and clinical adoptive transfer of sequential populations of well characterized melanoma specific T cell clones could help systematically define the specific antigenic targets and lymphocyte characteristics associated with T cell persistence, therapeutic efficacy, and toxicity. To overcome the significant obstacle in isolating multiple clonal variants for use in cancer therapy, we recently described a novel T cell isolation platform to rapidly obtain antigen specific T cell clones under GMP standards from the peripheral blood of patients. The methodology utilizes high throughput cytokine gene expression profiling to identify rare antigen specific T cells within heterogeneous cell populations. The technology is the subject of U.S. Patent Application (12/866,919) and was evaluated by the NCI Technology Advisory Group in 2011 and voted as one of NCIs top intramural inventions with respect to significance, innovation, and commercial potential (http://sbir.cancer.gov/funding/contracts/311.asp).This work has also resulted in a federal technology transfer award in 2011 (Recipient: Kammula). The versatility of the rapid cloning technology has allowed us to isolate a variety of CD8+ T cell clones that have specificity for common tumor antigens including gp100, MART, tyrosinase, NY-ESO, MAGE, and mesothelin. Further molecular characterization of these cells revealed that the stoichiometric production of IL-2 and IFN-g mRNA by these CD8+ T cells could further define a highly proliferative central memory (TCM) population. In the initial iteration of our research strategy, we translated these novel in vitro findings to a first-in-human clinical trial in which we adoptively transferred gp100 specific effector clones derived from TCM parental cells to patients with metastatic melanoma (NCI 08-C-0104). Analysis of this trial has revealed that these clones were able to target skin melanocytes in an autoimmune fashion, persist long term, and reacquire TCM attributes after transfer. This represents the first evidence in humans that TCM-derived effector clones can exert potent antigen specific effector function and also undergo self renewal to repopulate the memory pool after adoptive transfer. These findings have direct implications on the selection of T cells for future adoptive immunotherapy efforts and a manuscript is currently in revision for Science Translational Medicine. The methodology for isolation of tumor specific central memory cells is the subject of a second U.S. Patent Application (PCT/US2011/47719).